US 6844157 B2
The invention relates to a method for detecting microorganisms in a sample by means of a nucleic acid probe. Conventional detection methods are, for example, the in-situ hybridization of microorganisms with fluorescence-labeled oligonucleotide probes (fluorescent in-situ hybridization). A disadvantage of said method is that an epifluorescence microscope is required for evaluating the results. According to the invention, the disadvantages of the in-situ hybridization method are overcome by hybridizing the microorganisms to be detected in a sample with a specific nucleic acid probe, removing non-hybridized nucleic acid probe molecules, separating and then detecting and optionally quantifying the hybridized nucleic acid probe molecules.
1. A method of detecting microorganisms in a sample
a) fixing the microorganisms contained in the sample;
b) incubating the fixed microorganisms with detectable nucleic acid probe molecules which are capable of hybridizing to a target nucleic acid of the microorganism to be detected so as to form a complex between the detectable nucleic acid probe molecules and the target nucleic acid;
c) removing nonhybridized nucleic acid probe molecules from the fixed microorganisms;
d) incubating the fixed microorganisms with a separation solution at a temperature at or above 80° C. and up to 100° C. which denatures the detectable nucleic acid probe molecules from the target nucleic acid, yielding separated nucleic acid probe molecules wherein the separation solution is selected from the group consisting of water, DMSO, 1×SSC and 0.001-0.01 M Tris/HCl, pH 9.0+/−2.0; and
e) detecting the separated nucleic acid probe molecules, wherein the presence of the separated nucleic acid probe molecules correlates with the presence of the microorganism in the sample, and wherein the detection signal associated with the separated nucleic acid probe molecules is greater than that with separated nucleic acid probe molecules obtained by incubation in a formamide separation solution.
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a) fluorescence markers,
b) chemoluminescence markers,
c) radioactive markers,
d) enzymatically active group,
f) nucleic acid detectable by hybridization.
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19. A method of detecting microorganisms in a sample
a) incubating a sample comprising fixed microorganisms with detectable nucleic acid probe molecules which are capable of hybridizing to a target nucleic acid of the microorganism to be detected so as to form a complex between the detectable nucleic acid probe molecules and the target nucleic acid of the microorganism;
b) removing nonhybridized nucleic acid probe molecules from the fixed microorganisms;
c) incubating the fixed microorganisms with a separation solution at a temperature at or above 80° C. and up to 100° C. which denatures the nucleic acid probe molecules from the target nucleic acid, yielding separated nucleic acid probe molecules, wherein the separation solution is selected from the group consisting of water, DMSO, 1×SSC and 0.001-0.01 M Tris/HCl, pH 9.0+/−2.0; and
d) detecting the separated nucleic acid probe molecules, wherein the presence of the separated nucleic acid probe molecules correlates with the presence of the microorganism in the sample, and wherein the detection signal associated with the separated nucleic acid probe molecules is greater than that with separated nucleic acid probe molecules obtained by incubation in a formamide separation solution.
20. The method of
21. The method of
This application is a continuation under 35 U.S.C. 111(a) of International Application No. PCT/EP00/03989 filed May 4, 2000 and published as WO 00/68421 on Nov. 16, 2000, which claims priority under 35 U.S.C. 119 from German Application No. 199 21 281.3 filed May 7, 1999, and German Application No. 199 36 875.9 filed Aug. 5, 1999, all of which applications are incorporated herein by reference.
For many decades, microorganisms could be identified only after prior culturing of the microorganisms and amplification accordingly. In the case of viruses, the microorganisms are cultured on the respective host organism; in the case of bacteria, fungi and single-cell algae, the cells are cultured in culture media. Media and conditions which should largely prevent selective detection of certain groups are selected for detecting the number of viable microorganisms in a certain specimen. For example, pure cultures of individual bacterial colonies are prepared and then identified on the basis of phenotypic features such as their morphology and metabolic pathways. However, identification of microorganisms after prior culturing is associated with two important disadvantages. First, studies on a wide variety of environmental specimens have shown that only 0.1% to 14% of all bacteria can be cultured at the present time. Secondly, it has been demonstrated that extreme population shifts can occur in culturing, i.e., certain groups of bacteria are preferred in the laboratory and others are discriminated against.
This means not only that a majority of the bacteria in environmental samples cannot be identified, but also that those bacteria which are identified usually represent a distorted image of the true population structures. This results in incorrect estimates of the population ratios with respect to identification and quantification of bacteria.
At the beginning of the '90s, a method of in situ hybridization with fluorescence-labeled oligonucleotide probes was developed and has been used successfully in many environmental samples (Amann et al. (1990), J. Bacteriol. 172:762). This method, which is known as “FISH” (fluorescence in situ hybridization) is based on the fact that the ribosomal RNA (rRNA) present in each cell includes both highly conserved sequences, i.e., those with a low specificity, and less conserved sequences, i.e., genus-specific and species-specific sequences. By the middle of the '80s, it had been demonstrated that the sequences of the 16S— and 23S-rRNA can be used for identification of microorganisms (Woese (1987), Microbiol. Reviews 51:221; De Long et al. (1989), Science 243:1360). In the case of the FISH method, fluorescence-labeled gene probes whose sequences are complementary to a certain region on the ribosomal target sequences are introduced into the cells. The probe molecules are usually 16- to 20-base-long, single-stranded deoxyribonucleic acid fragments, and they are complementary with a target range which is specific for a certain species or genus of bacteria. If the fluorescence-labeled gene probe finds its target sequence in a bacterial cell, it binds to it, and the cells can be detected on the basis of their fluorescence in a fluorescence microscope.
It has been demonstrated that up to 90% of a total bacterial population can be detected by in situ hybridization with fluorescence-labeled probes. Therefore, this method represents a significant improvement in comparison with the state of the art, which has made it possible to detect a maximum of 14% of the bacterial population in an environmental specimen. In addition, the method of in situ hybridization with fluorescence-labeled probes makes it possible to determine the active portion of a population by determining the ratios of a probe directed against all bacteria relative to the dry weight. Finally, this method makes it possible to visualize bacteria directly at their site of action, which thus makes it possible to detect and analyze interactions between different bacterial populations.
Within recent years, the technique of in situ hybridization with fluorescence-labeled probes has been tested and used successfully with a wide variety of environmental samples. Thus, by using gene probes in soil, protozoa, biofilms, air, lake water, biologically activated filters and wastewater from sewage treatment facilities, it has been possible to investigate the respective bacterial populations and identify new types of bacteria. The main point of emphasis here has been analysis of the bacterial populations in wastewater purification. Trickle filters, room filters and activated sludge installations have been investigated, along with secondary clarification ponds and corresponding receiving streams (Snaidr et al. (1997), Appl. Environ. Microbiol. 63:2884). It has been shown by means of the in situ hybridization technique that when activated sludge flora are detected by culturing, there is a culturing shift (Wagner et al. (1993), Appl. Environ. Microbiol. 59:1520). Therefore, methods that depend on culturing give a highly distorted view of the composition and dynamics of the microbial biocenosis. The significance of bacteria, which play only a subordinate role in activated sludge but are well adapted to the given culturing conditions, is dramatically overestimated due to this distortion of the actual conditions within the bacterial population which occurs as a function of the medium. It has thus been demonstrated that because of such a culturing artefact, the role of the bacterial genus Acinetobacter as a biological agent for removing phosphates in wastewater purification has been completely misappraised.
Although in situ hybridization with the newly developed fluorescence-labeled gene probes permits a rapid and accurate analysis of bacteria populations in wastewater, it has not yet been successful in practice. Reasons for this include the high cost of acquisition of the necessary technical equipment such as a fluorescence microscope, the demand for qualified personnel, who must be available to perform the tests and analyze the results, as well as the resulting low number of possible reference measurements. In addition, it is very time-consuming to count the bacterial populations thus detected (quantification). Moreover, such counts require high empirical values because it is necessary to differentiate between true signals (probe binding has in fact occurred) and false signals (autofluorescence, no cells).
Therefore, the object of the present invention is to make available a method with which microorganisms can be detected and optionally quantified, preferably without prior culturing.
Another important object of the present is to provide a method of detecting microorganisms, to be carried out under conditions which guarantee that the measurement results will not be influenced in a negative sense by detection and quantification, if desired.
Furthermore, another object of the present invention is to provide a method for detection of microorganisms which can be carried out more rapidly and in a more environmentally friendly manner and is easier to handle than the methods described in the state of the art.
According to this invention, these objects are achieved by a method for detecting microorganisms in a sample by means of a nucleic acid probe, where this method includes the following steps:
Within the context of the present invention, the term “fixation” of microorganisms should be understood to refer to a treatment with which the coat or shell surrounding the respective microorganism is made so permeable that the nucleic acid probe having the optionally covalently bonded labeling can penetrating through the coat to reach the target sequences in the interior of the cell. The coat may be a lipid coat surrounding a virus, for example, the cell a wall of a bacterium or the cell membrane of a single-cell microorganism. A paraformaldehyde solution with a low percentage concentration is generally used for the fixation. If, in the individual case, the protective coat surrounding a microorganism cannot be made penetrable with a paraformaldehyde solution, those skilled in the art will be familiar with sufficient other measures leading to the same result. Such measures would include for example, ethanol, methanol, mixtures of these alcohols with paraformaldehyde, enzymatic treatments, ultrasonic treatment, etc.
In the case of a nucleic acid probe in the sense of this invention, it may be a DNA or RNA probe, which usually comprises between 12 and 1000 nucleotides, preferably between 12 and 100 or 15 and 50 nucleotides, especially preferably between 17 and 25 nucleotides. The nucleic acid probe is selected so that there is a sequence complementary to it in the microorganism to be detected or in the group of microorganisms to be detected. In the case of a probe with only approximately 15 nucleotides, the requirement of complementarity must be met over 100% of the sequence; in the case of oligonucleotides with more than 15 nucleotides, one or more mismatched sites are allowed. However, it must be guaranteed that the nucleic acid probe molecule will in fact hybridize with the target sequence under moderate and/or stringent hybridization conditions. Moderate conditions in the sense of this invention would include, for example, 0% formamide in a hybridization buffer such as that described in Example 1. Stringent conditions in the sense of this invention would include, for example, 20% to 80% formamide in the buffer described in section 5.2 of Example 1.
The duration of hybridization is usually between 10 minutes and 12 hours. Hybridization preferably takes place for about two hours. The hybridization temperature is preferably between 44° C. and 48° C., especially preferably 46° C., where the parameter of the hybridization temperature as well as the concentration of salts and detergents in the hybridization solution can be optimized as a function of the probe or probes, in particular their length(s) and the degree of complementarity with the target sequence in the cell to be detected. Those skilled in the art will be familiar with the relevant calculations in this regard.
In the scope of the method according to this invention, a typical hybridization solution would have a salt concentration of 0.1 M to 1.5 M, preferably 0.9 M, with the salt preferably being sodium chloride. In addition, the hybridization buffer usually includes a detergent such as sodium dodecyl sulfate (SDS) in a concentration of 0.001 to 0.1%, preferably in a concentration of 0.01%, and Tris/HCl in a concentration range of 0.001-0.1 M, preferably in a concentration of 0.02 M. The pH value of Tris/HCl is usually between 6 and 10, but a pH of approximately 8.0 is preferred. As mentioned above, the hybridization solution may also contain between 0% and 80% formamide, depending on the degree of stringency desired or required.
If possible, the nucleic acid probe should be present in the hybridization buffer in an amount of 15 ng to 1000 ng, with this amount preferably being in 80 μL hybridization solution. The probe concentration especially preferably amounts to 125 ng/80 μL hybridization solution.
After successful hybridization, the unhybridized and excess probe molecules should be removed, which is usually accomplished by means of a traditional washing solution or washing buffer. The washing buffer usually contains 0.001% to 0.1% of a detergent such as SDS, with a concentration of 0.01% being preferred, and Tris/HCl in a concentration of 0.001 to 0.1 M, preferably 0.02 M, with the pH value of Tris/HCl being in the range of 6.0 to 10.0, preferably 8.0. In addition, the washing buffer usually contains NaCl, but the concentration amounts to between 0.003 M and 0.9 M, preferably between 0.01 M and 0.9 M, depending on the stringency required. In addition, the washing solution may also contain EDTA, in which case the concentration is preferably 0.005 M.
The unbound probe molecules are usually “washed out” at a temperature in the range of 44° C. to 52° C., preferably between 46° C. and 50° C., and especially preferably at 48° C. for a period of 10 to 40 minutes, preferably for 20 minutes.
After successful in situ hybridization, followed by removal of the unhybridized nucleic acid probe molecules by means of the washing step described above, the hybridized probe molecules are isolated for detection, and if desired, the hybridized probe molecules are quantified.
For this extraction step, extraction agents which guarantee denaturing of the probe from the target sequence at a suitable temperature without damaging the probe molecule to any significant extent are suitable for this extraction step. The latter is desirable in particular in order not to have a negative effect on the measurement results in the subsequent detection and quantification. The preferred separation solution or buffer for this purpose is water, i.e., distilled or double-distilled H2O or weakly buffered water, i.e., for example, Tris/HCl in a concentration range of 0.001 M to 1.0 M, especially preferably in a concentration of 0.01 M, with the pH of Tris/HCl being between 7.0 and 11.0, preferably 9.0. In addition, DMSO (dimethyl sulfoxide) and 1×SSC, pH 10.0 (+/−2.0) are also suitable extraction agents for use according to this invention, in which case 1×SSC is suitably prepared by diluting a 20×SSC stock solution (175.2 g NaCl, 88.2 g sodium citrate, adding water to a total of one liter).
Separation of the probe molecules is usually performed for a period of 5 to 30 minutes, preferably 15 minutes. Probe extraction is performed in a temperature range of 50 to 100° C., preferably 100° C., especially preferably at approximately 80° C. In any case, an attempt should be made to perform the extraction at a temperature which is effective but at the same time guarantees gentle separation of the probe molecules. Since the probes are less affected by the extraction treatment as the temperature is lower, a temperature of <100° C., especially <90° C. is preferred.
In a comparion experiment, 4.8×106 Burkholderia cepacia cells were hybridized with two fluorescence-labeled oligonucleotide probes (each 2.5 μL BET42a-Cy3 or NonEUB338-Cy3) by means of the fast FISH method according to this invention. Probe extraction was performed for 15 minutes at 80° C. with 110 μL separation solution, comparing double-distilled H2O, 0.01 M Tris/HCl, pH 9.0, 1×SSC, pH 10.0, DMSO and formamide with one another with regard to the measurement signal, i.e., the fluorescence intensity. Although good to very good results were achieved with water, 0.01 M Tris/HCl, 1×SSC and DMSO, no comparable measurement signal comparable to probe extraction with the other separation solutions could be obtained when using formamide. Instead, the signal that was obtained with formamide as the extraction solution was lower by a factor of at least 10 than the intensities measured with double-distilled H2O or 0.01 M Tris/HCl, for example. At a separation temperature of 100° C. (i.e., an extraction agent temperature of 100° C.), the difference in the measurement signals was even more pronounced between formamide on the one hand and double-distilled H2O or 0.01 M Tris/HCl on the other hand. These observations can in all probability be attributed to an even greater negative effect of formaldehyde on the probe molecules at higher temperatures.
On the basis of these observations, double-distilled H2O or 0.01 M Tris/HCl in particular is preferred as the extraction agent in comparison with formamide, which is usually recommended as the extraction agent in the state of the art. Due to the fact that formamide is not used in the extraction step, not only is the resulting treatment less damaging, but also there are additional advantages with regard to the potential hazard involved with formamide, which is a substance of toxicity class 3. In addition, the use of formamide is much more cost-intensive in comparison with water or weakly buffered water.
The respective nucleic acid probe is selected as a function of the microorganism to be detected. For example, if only microorganisms of the species Streptococcus salivarius are to be detected, but microorganisms of the species Streptococcus thermophilus are not to be detected, then those skilled in the art will select a suitable sequence which occurs in Streptococcus salivarius but does not occur in Streptococcus thermophilus. These sequences are typically selected from the 16S-rRNA or the 23S-rRNA. However, if all the bacteria of the genus Streptococcus are to be detected, then a sequence which is found in common in both Streptococcus salivarius and Streptococcus thermophilus as well as additional species of the genus Streptococcus would be selected. Many examples of such sequences have already been published in the literature (see, for example, Beimfohr et al. (1993), System Appl. Microbiol. 16:450). The nucleic acid probe may be complementary to a chromosomal or episomal DNA, but it may also be complementary to an mRNA or an rRNA of the microorganism to be detected. The preferred nucleic acid probes for selection are those which are complementary to a range occurring in the respective microorganism to be detected in more than one copy. The sequence to be detected preferably occurs 500 to 100,000 times per cell, especially preferably in 1000 to 50,000 times. This is another reason why it is especially preferable for the nucleic acid probe to be complementary to an rRNA: ribosomal RNA is part of ribosomes which are present many thousand times in each active cell because they are protein synthesis molecules.
According to this invention, the nucleic acid probe is incubated with the fixed microorganism in the above-mentioned sense, in order to thereby permit penetration of the nucleic acid probe molecules into the microorganism and hybridization of nucleic acid probe molecules with the nucleic acids of the microorganisms. Following that, nonhybridized nucleic acid probe molecules are removed by the usual washing steps. In contrast with the traditional FISH method, however, now the hybridized nucleic acid probe molecules are not left in situ, i.e., in the respective microorganism, but instead they are isolated again from the nucleic acid to be detected and are separated from cellular constituents, detected and optionally quantified. The prerequisite for this is that the nucleic acid probe molecule used according to this invention can be detected. This detectability can be ensured, for example, by covalent bonding of the nucleic acid probe molecule to a detectable marker. Detectable markers usually include fluorescent groups such as Cy-2, Cy-3 or Cy-5, FITC, CT, TRITC or Fluos-Prime, all of which will be very familiar to those skilled in the art. For the sake of thoroughness, Table 1 below lists a few markers, their properties and sources for ordering them.
When using fluorescent markers, the method according to invention is also referred to below as a “fast FISH” method, based on the terminology used for the above-mentioned FISH method.
As an alternative, chemoluminescent groups or radioactive markers such as 35S, 32P, 33P, 125I may be used. However, detectability may also be ensured by coupling the nucleic acid probe molecule with an enzymatically active molecule such as alkaline phosphatase, acid phosphatase, peroxidase, horseradish peroxidase, β-D-galactosidase or glucose oxidase. For each of these enzymes, there are a number of known chromogens which can be reacted instead of the natural substrate and can be converted either to colored or fluorescent products. Examples of such chromogens are given in Table 2 below.
Finally, it is possible to design the nucleic acid probe molecules so that another nucleic acid sequence which is suitable for hybridization is present at its 5′- or 3′-end. This nucleic acid sequence in turn includes approximately 15 to 1000 nucleotides, preferably 15 to 50 nucleotides. The second nucleic acid range may in turn be recognized by oligonucleotide probes which can be detected by one of the means mentioned above.
Another possibility is coupling the detectable nucleic acid probe molecules with a haptene. After separation of the nucleic acid probe molecules from the target nucleic acid, the nucleic acid probe molecules which are then separate can be brought in contact with detectable antibodies that recognize the haptene. A known example of such a haptene is digoxigenin or its derivatives. Those skilled in the art will be familiar with many additional possibilities in addition to those given in the examples for detecting and quantifying an oligonucleotide that is used for hybridization.
The plurality of possible markings also permits simultaneous detection of two or more different overlapping or nonoverlapping populations. For example, by using two different fluorescence markers, it is possible to detect Streptococcus salivarius as well as Streptococcus thermophilus or Streptococcus salivarius in addition to the total streptococcal population.
The microorganism to be detected by the method according to this invention may be a prokaryotic or eukaryotic microorganism. In most cases, it is desirable to detect single-cell microorganisms. Relevant microorganisms include mainly yeasts, bacteria, algae and fungi.
In an especially preferred embodiment of the invention, the microorganism is a member of the genus Salmonella.
The method according to this invention may be used in a variety of ways. For example, it is suitable for investigating environmental samples for the presence of certain microorganisms. These environmental samples can be obtained from water, soil or air. Normally no prior culturing is necessary for detection of certain bacteria in environmental samples.
Another important application for the method according to this invention is for inspection of food samples. In preferred embodiments, the food samples are taken from milk or milk products (yogurt, cottage cheese, hard cheese, butter, buttermilk), drinking water, beverages (juice, lemonade, beer), bakery goods or meat products. Under some circumstances, prior culturing may be desirable or even required for detection of microorganisms in foods. For example, for detection of a single organism of Salmonella in 25 mL milk, the milk must first be cultured for a period of time to then find one or more Salmonella in the sample volume with a statistical reliability.
The method according to this invention can also be used for testing medical samples. It is suitable for testing tissue samples such as biopsy material taken from the lung, from a tumor or inflammatory tissue, from secretions such as perspiration, saliva, sperm and secretions from the nose, urethra or vagina or for urinalysis and stool specimens.
Another area of application of the method according to this invention is for testing wastewater such as activated sludge, putrefactive sludge or anaerobic sludge. In addition, it is suitable for analyzing biofilms in industrial installations as well as naturally occurring biofilms or biofilms formed in wastewater purification. Finally, it is also suitable for testing and for quality control of pharmaceutical and cosmetic products such as creams, ointments, tinctures, liquids, etc.
The method according to this invention represents a possibility of establishing in situ hybridization for cell identification and optionally quantification in practice. The required equipment would be limited to the acquisition of a fluorometer (max. approximately DM 18,000), for example, when using fluorescent molecular probes. In contrast with that, an epifluorescence microscope, which is suitable for carrying out the traditional FISH method and with which sufficiently good in situ hybridization results can be achieved, is in a price range of approximately 100,000 DM. Furthermore, when using Cy-5 labeled probes, for example, the epifluorescence microscope must also be equipped with a high-quality CCD camera (price between DM 30,000 and DM 50,000). For this reason, the method according to this invention is a much less expensive measurement method than the time-consuming quantification using an epifluorescence microscope. Moreover, the ongoing cost of the method according to this invention using a fluorometer is much lower than that of the traditional method using epifluorescence microscopy. This is due mainly to the fact that the mercury high pressure lamps (each DM 450) of the epifluorescence microscope must be replaced at the latest after 100 hours of operation for reasons of quality and safety. The enormously time-consuming process of counting specifically labeled cells under the microscope thus leads to great wear and tear on the lamp. The xenon arc lamp of a fluorometer (DM 3000) has a lifetime of one to three years even with extensive use of the instrument. The cost of the personnel required to perform the measurements represents an additional cost factor. Although a quantitative analysis of an environmental sample by means of the traditional method requires several days, especially when multiple probes are used, the method according to this invention should handle this job within a few hours. For hybridization and extraction, a period of three hours would be needed, whereas quantification in a fluorometer would take only a few minutes. Quantification could also be performed by untrained personnel, whereas with the traditional method, visual quantification requires the skills of a specialist.
Thus, the present invention makes available a method which can be carried out much more easily and more rapidly in comparison with the traditional methods and is also safer for the environment.
Although this invention has been described essentially with respect to fluorescence-labeled probe molecules, it is self-evident that the above-mentioned advantages are also obtained when using other markers.
According to this invention, a kit is made available for carrying out the method for detecting microorganisms in a sample. The contents of such a kit will depend essentially on the nature of the microorganism to be detected. It will include as the most important component a nucleic acid probe which is specific for the respective microorganisms to be detected as well as another nucleic acid probe with which a negative control can be performed. In addition, it includes a hybridization buffer and optionally a lysis buffer. The choice of the hybridization buffer will depend primarily on the length of the nucleic acid probes used. Thus, as those skilled in the art will be aware, less stringent conditions are necessary for hybridization of a nucleic acid probe which is 15 nucleotides long than for hybridization of a probe 75 nucleotides long. Examples of hybridization conditions are given, e.g., by Stahl & Amann (1971) in Stackebrandt and Goodfellow (eds.), Nucleic Acid Techniques in Bacterial Systematics; John Wiley & Sons Ltd., Chichester, UK.
The composition of the lysis buffer also depends on the respective microorganism. Thus, slightly different conditions are necessary for lysis of viral coats, cell walls of gram-positive or gram-negative bacteria, and cell membranes of yeasts or algae, but these conditions can be determined readily in the respective literature.
In a preferred embodiment, the kit according to this invention contains specific probes for detection of bacteria of the genus Salmonella. In an especially preferred embodiment, the nucleic acid probe molecule for specific detection of a microorganism is the nucleic acid sequence:
The following illustrations and the examples are presented to illustrate this invention and should not be interpreted as restricting the scope in any way.
The mixture of cells, some of which have bound nucleic acid probe molecules of type A, is put through a separation step. Then the nucleic acid probe molecules of type A separated in this way are quantified.
Batch with Salmonella:
Batch without Salmonella:
Salm63-Cy3 detects Salmonella species, and it represents the background with the Salmonella-specific probe because no Salmonella were present in this batch (control 2).
Detection of bacteria of the genus Salmonella in milk
1. General Description:
The method described below, which is referred to as the “SalmoQuick method” according to this invention, is used for qualitative analysis of bacteria of the genus Salmonella in foods on the basis of the method according to this invention. Salmonella can be identified in 24 hours, so this yields a considerable speed advantage in comparison with conventional methods which take between 5 and 14 days for an identification, depending on the taxonomic accuracy.
2. Basic Principle:
Salmonella in milk is detected specifically by fluorescence-labeled oligonucleotide probes directed against rRNA. After suitably stringent washing steps, bound probes are released from their target cells in the bacteria and then are quantified in a fluorometer. A conclusion regarding whether or not Salmonella is present in milk can obtained on the basis of the height of the resulting fluorometer signal.
3. Brief Description:
The milk sample which is to be tested for the presence of Salmonella is incubated for several hours. This ensures that first, there are enough target sites for detection with probes due to the reproduction of any Salmonella that might be present in the milk, and secondly, only living Salmonella are identified. A shift in population due to incubation for several hours is not harmful, because not all bacteria need be identified and instead only the presence or absence of Salmonella is determined. After centrifugation and fixation of the cells, during which the cells are made accessible for the probes, the proteins which interfere with the subsequent hybridization can be removed sufficiently well by a lysis step. During the subsequent hybridization, the fluorescence-labeled oligonucleotide probes bind specifically to the rRNA or the bacteria of the genus Salmonella under sufficiently stringent conditions. The subsequent washing step ensures removal of the unbound probes. During another treatment procedure, the specifically bound probes are extracted from the cells. The fluorescence colors of these probes can then be quantified in a fluorometer. The height of the resulting signal provides information regarding whether or not Salmonella is present in the milk sample.
4. Technical Equipment:
4.1 Preparation of the Sample
The following equipment is needed to prepare a sample:
The following are needed for in situ hybridization:
The following material were used to process the sample, including cell fixation:
Can be prepared by adding 3 g paraformaldehyde to 30 mL double-distilled H2O heated to 60° C., adding 1 M NaOH by drops until the paraformaldehyde is dissolved completely, then adding 16.6 mL 3×PBS, cooling the solution to approximately 20° C., adjusting the pH with 1 M HCl to 7.2-7.4; sterile filtration of the finished PFA solution through a 0.2 μm filter (Millipore, Eschborn). The solution can be stored for approximately 1 week at 4° C.; freezing for several months is also possible.
5.2 In situ hybridization
The result of the experiment described above is illustrated in
Detection of bacteria of the genus Salmonella in 25 mL milk or 25 g milk powder
Batch of two milk samples of 25 mL each or 25 g for hybridization with the probes
Pour 225 mL portions into 1 L Erlenmeyer flask and 30 mL portions into 100 mL Erlenmeyer flask and autoclave for 15 minutes at 121° C.
Solutions used for PFA cell fixation:
This solution can be prepared by adding 3 g paraformaldehyde to 30 mL double-distilled H2O heated to 60° C., then adding 1 M NaOH by drops until the paraformaldehyde has dissolved completely, next adding 16.6 mL 3×PBS, cooling the solution to approximately 20° C., adjusting the pH with 1 M HCl to 7.2-7.4, and sterile filtration of the finished PFA solution through a 0.2 μm filter (Millipore, Eschborn). The solution can be stored for approximately one week at 4° C. It can also be stored frozen for several months.
17. Transfer 800 μL into each of two 1.5 mL Eppendorf reaction vessels for subsequent hybridization with Salm63-Cy3 and nonSalm-Cy3.
Use of the fast FISH technique for relative quantification of bacterial populations in activated sludge (on the example of PPx3)
A. Cell fixation:
This solution can be prepared by adding 3 g paraformaldehyde to 30 mL double-distilled H2O heated to 60° C., adding 1 M NaOH by drops until the paraformaldehyde has dissolved completely, then adding 16.6 mL 3×PBS, cooling the solution to approximately 20° C., adjusting the pH with 1 M HCl to 7.2-7.4, sterile filtration of the finished PFA solution through a 0.2 μm filter (Millipore, Eschborn). The solution can be stored for approximately one week at 4° C. It can also be stored frozen for several months.
Calculating the relative amount of PPx3 cells in the activated sludge tested: